ABSTRACT ETS family protein alterations (primarily ERG translocations) are present in >50% of human prostate cancers in Western men. We were the first to develop a robust GEM model of ERG-driven prostate cancer, revealing enhanced luminal differentiation and an expanded androgen receptor (AR) cistrome in tumors, suggesting a novel mechanism for ERG oncogenicity via chromatin programming (Chen, 2013). We and others subsequently established that ERG activates a luminal differentiation program in prostate organoids, human prostate cell lines and, by computational analysis of prostate cancer genomes, in clinical samples (Blee et al., 2018; Kron et al., 2017; Li et al., 2020b). Other ETS gain-of-function alterations e.g. ETV4 translocations (Li et al., 2020a) and ETS loss-of-function alterations e.g. ERF repressor mutations/deletions (Bose et al., 2017) also show this phenotype, as does FOXA1 (also amplified or mutated in prostate cancer) but with a contracted AR cistrome (Adams et al., 2019). Having demonstrated that luminal differentiation is a primary feature of multiple oncogenic ETS proteins (and FOXA1), our major goal during the next funding cycle is to understand how ERG activates this differentiation program and how this program results in an oncogenic phenotype. We will pursue three parallel lines of investigation. First, from our biochemical studies using purified full-length proteins and various DNA templates, we find that that ERG (and other ETS factors) cooperatively enhance AR DNA binding through allosteric effects via direct protein-protein interaction; biologically, this broadens the AR cistrome to include novel AR binding sites (Wasmuth et al., 2020). Aim 1 will expand this analysis to assess the role of FOXA1 on AR/ERG interactions. Second, we have built genetically defined prostate organoid models that recapitulate the luminal differentiation effect of ERG within a precisely defined time course. Using this system, we identified epigenetic changes that silence transcription of the basal epithelial master regulator p63, likely explaining the reduction in basal cells. Aim 2 will use lineage tracing, single cell analysis, and CRISPR screening to further elucidate how ERG expands the number of luminal cells and initiates oncogenic transformation. Third, we showed that another oncogenic ETS protein (ETV4) also drives luminal differentiation and is sufficient, alone, to initiate prostatic intraepithelial neoplasia (PIN). Remarkably, these luminal epithelial cells acquire exquisite dependence on AR for survival (in contrast to normal luminal epithelial cells) and consequently display enhanced sensitivity to androgen deprivation therapy (ADT). Aim 3 will explore mechanisms underlying this shift to cell-intrinsic AR dependence by examining changes in the AR cistrome and transcriptome following luminal-specific AR ablation (by genetic deletion) versus systemic androgen deprivation through surgical castration (impairs AR function in prostate st...